Methods of purifying anti-interleukin-13 antibodies

ABSTRACT

The invention relates to a pharmaceutical composition comprising an interleukin-13 antibody, more particularly a monoclonal interleukin-13 antibody, especially a human interleukin-13 monoclonal antibody, to a process for purifying said antibody and to the use of said composition in treating interleukin-13 related disorders, such as asthma, atopic dermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonary disease, scleroderma, inflammatory bowel disease and Hodgkin&#39;s lymphoma, particularly asthma.

The invention relates to a pharmaceutical composition comprising aninterleukin-13 antibody, more particularly a monoclonal interleukin-13antibody, especially a human interleukin-13 monoclonal antibody, to aprocess for purifying said antibody and to the use of said compositionin treating interleukin-13 related disorders, such as asthma.

Interleukin (IL)-13 is a 114 amino acid cytokine with an unmodifiedmolecular mass of approximately 12 kDa. IL-13 is most closely related toIL-4 with which it shares 30% sequence homology at the amino acid level.The human IL-13 gene is located on chromosome 5q31 adjacent to the IL-4gene [McKenzie, A. N. et al., J Immunol, 1993. 150(12), 5436-5444;Minty, A. et al., Nature, 1993. 362(6417), 248-50].

Although initially identified as a Th2 CD4+ lymphocyte derived cytokine,IL-13 is also produced by Th1 CD4+ T-cells, CD8+ T lymphocytes NK cells,and non-T-cell populations such as mast cells, basophils, eosinophils,macrophages, monocytes and airway smooth muscle cells.

IL-13 has been linked with a number of diseases, in particular, diseaseswhich are caused by an inflammatory response. For example,administration of recombinant IL-13 to the airways of naivenon-sensitised rodents was shown to cause many aspects of the asthmaphenotype including airway inflammation, mucus production and airwayshyper-responsiveness (AHR) [Wills-Karp, M. et al., Science, 1998.282(5397), 2258-2261; Grunig, G. et al., Science, 1998. 282(5397),2261-2263; Venkayya, R., et al., Am J Respir Cell Mol Biol, 2002. 26(2),202-208; Morse, B. et al., Am J Physiol Lung Cell Mol Physiol, 2002.282(1), L44-49]. A similar phenotype was observed in a transgenic mousein which IL-13 was specifically overexpressed in the lung. In thismodel, more chronic exposure to IL-13 also resulted in fibrosis [Zhu, Z.et al., J Clin Invest, 1999. 103(6), 779-788].

A number of genetic polymorphisms in the IL-13 gene have also beenlinked to allergic diseases. In particular, a variant of the IL-13 genein which the arginine residue at amino acid 130 is substituted withglutamine (Q130R) has been associated with bronchial asthma, atopicdermatitis and raised serum IgE levels [Heinzmann, A. et al., Hum MolGenet, 2000. 9(4), 549-559; Howard, T. D. et al., Am J Hum Genet, 2002.70(1), 230-236; Kauppi, P. et al., Genomics, 2001. 77(1-2), 35-42;Graves, P. E. et al., J Allergy Clin Immunol, 2000. 105(3), 506-513].This particular IL-13 variant is also referred to as the Q110R variant(arginine residue at amino acid 110 is substituted with glutamine) bysome groups who exclude the 20 amino acid signal sequence from the aminoacid count.

IL-13 production has also been associated with allergic asthma [van derPouw Kraan, T. C. et al., Genes Immun, 1999. 1(1), 61-65] and raisedlevels of IL-13 have been measured in human subjects with atopicrhinitis (hay fever), allergic dermatitis (eczema) and chronicsinusitis.

Aside from asthma, IL-13 has been associated with other fibroticconditions. Increased levels of IL-13, up to a 1000 fold higher thanIL-4, have been measured in the serum of patients with systemicsclerosis and in broncho-alveolar lavage (BAL) samples from patientsaffected with other forms of pulmonary fibrosis [Hasegawa, M. et al., JRheumatol, 1997. 24(2), 328-332; Hancock, A. et al., Am J Respir CellMol Biol, 1998. 18(1), 60-65].

It has been demonstrated that overexpression of IL-13 in the mouse lungcaused emphysema, elevated mucus production and inflammation, reflectingaspects of human chronic obstructive pulmonary disease (COPD) [Zheng, T.et al., J Clin Invest, 2000. 106(9), 1081-1093].

It has been proposed that IL-13 may also play a role in the pathogenesisof inflammatory bowel disease [Heller, F. et al., Immunity, 2002. 17(5),629-38] and raised levels of IL-13 have been detected in the serum ofsome Hodgkin's disease patients when compared to normal controls[Fiumara, P. et al., Blood, 2001. 98(9), 2877-2878].

IL-13 inhibitors are also believed to be therapeutically useful in theprevention of tumour recurrence or metastasis [Terabe, M. et al., NatImmunol, 2000. 1(6), 515-520]. Inhibition of IL-13 has also been shownto enhance anti-viral vaccines in animal models and may be beneficial inthe treatment of HIV and other infectious diseases [Ahlers, J. D. etal., Proc Natl Acad Sci USA, 2002. 99(20), 13020-13025].

An antibody directed approach to IL-13 inhibition has been described.For example, WO 2005/007699 (Cambridge Antibody Technology Limited)describes a series of human anti-IL-13 antibody molecules which areshown to neutralise IL-13 activity and which are claimed to be ofpotential use in the treatment of IL-13 related disorders.

According to a first aspect of the invention there is provided apharmaceutical composition comprising an IL-13 antibody and one or morepharmaceutically acceptable excipients buffered to a pH of 4.5-6.0 withacetate buffer.

It is commonly known that antibody purification procedures typicallyrequire a number of separation techniques, such as chromatographyseparations (e.g. Protein A chromatography, ion exchange chromatographyand the like). A consequence of this manner of separation requires theuse of a number of differing buffers. For example, the antibodypurification procedure described in WO 2004/076485 requires the use of50 mM glycine/glycinate pH 8.0 for Protein A chromatography, 50 mMTrisHCl pH 8.0 and 20 mM sodium phosphate pH 6.5 for Q-Sepharosechromatography, and 25 mM TrisHCl pH 8.6 for DEAE-sepharosechromatography.

By contrast, the present invention requires the use of a single acetatebuffer at a fixed concentration and predetermined pH present within thecomposition of the invention which has the advantage of being presentthroughout all IL-13 antibody purification steps. Thus, the use of thisbuffer, not only at the beginning of the purification process, butthroughout the entire purification process, therefore results in areduction of processing time, cost and an increase in product yield.

It will be appreciated that references to “antibody” include referencesto an immunoglobulin whether natural or partly or wholly syntheticallyproduced. The term also covers any polypeptide or protein comprising anantigen binding domain.

Antibody fragments which comprise an antigen binding domain aremolecules such as Fab, scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB2188638A or EP-A-239400, and a large body ofsubsequent literature.

Alternatively, novel VH or VL regions carrying CDR-derived sequences maybe generated using random mutagenesis of one or more selected VH and/orVL genes to generate mutations within the entire variable domain. Such atechnique is described by Gram et al., PNAS USA, 1992. 89, 3576-3580,who use error prone PCR. Another method which may be used is to directmutagenesis to CDR regions of VH and VL genes. Such techniques aredisclosed by Barbas et al., PNAS USA 1994. 91, 3809-3813 and Schier etal., J. Mol. Biol. 1996. 263, 551-567.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving an antigen-binding domain with the required specificity. Thus,this term covers antibody fragments and derivatives, including anypolypeptide comprising an immunoglobulin binding domain, whether naturalor wholly or partially synthetic. Chimeric molecules comprising animmunoglobulin binding domain, or equivalent, fused to anotherpolypeptide are therefore included. Cloning and expression of chimericantibodies are described in EP-A-0120694 and EP-A-0125023, and a largebody of subsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann et al., [2001;Antibody Engineering, Springer Laboratory Manuals]. Phage display,another established technique for generating specific binding membershas been described in detail in many publications such as Kontermann etal. (supra) and W092/01047. Transgenic mice in which the mouse antibodygenes are inactivated and functionally replaced with human antibodygenes while leaving intact other components of the mouse immune system,can be used for isolating human antibodies to human antigens. Ribosomedisplay, a cell free translation technology which introduces mutationsinto known gene sequences, may also be used to generate and/or optimisespecific binding members [Hanes and Plückthun PNAS USA, 1994. 94,4937-4942; He and Taussig Nucleic Acids Res. 1997. 25, 5132-5134;Schaffitzel et al., J. Immunol. Methods, 1999. 231, 119-135; He et al.,J. Immunol. Methods, 1999. 231, 105-117; He et al., Methods Mol. Biol.2004. 248, 177-189].

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.,J. Mol. Biol. 2000. 296, 57-86 or Krebs et al., Journal of ImmunologicalMethods 2001. 254, 67-84.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment [Ward, E. S. et al., Nature, 1989. 341, 544-546; McCafferty etal., Nature, 1990. 348, 552-554; Holt et al., Trends Biotechnol. 2003.21(11), 484-490] which consists of a VH or a VL domain; (v) isolated CDRregions; (vi) F(ab') 2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site [Bird etal., Science, 1988. 242, 423-426; Huston et al., PNAS USA, 1988. 85,5879-5883]; (viii) bispecific single chain Fv dimers (PCT/US92/09965)and (ix) “diabodies”, multivalent or multispecific fragments constructedby gene fusion [W094/13804; P. Holliger et al., Proc. Natl. Acad. Sci.USA, 1993. 90, 6444-6448]. Fv, scFv or diabody molecules may bestabilised by the incorporation of disulphide bridges linking the VH andVL domains [Y. Reiter et al., Nature Biotech., 1996. 14, 1239-1245].Minibodies comprising a scFv joined to a CH3 domain may also be made [S.Hu et al., Cancer Res., 1996. 56, 3055-3061].

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways[Holliger and Winter Current Opinion Biotechnol. 1993. 4, 446-449], e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. Examples of bispecificantibodies include those of the BiTE technology in which the bindingdomains of two antibodies with different specificity can be used anddirectly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (W094/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against IL-13, then a library can be made where the other armis varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering[Ridgeway et al., Protein Eng., 1996. 9, 616-621].

It will be appreciated that references to “an IL-13 antibody” includereferences to a whole antibody or antibody fragment which is capable ofneutralising naturally occurring IL-13 at a concentration of less than500 nM by following the assays as set forth in Examples 2-10 and 25 ofWO 2005/007699.

Preferably, the IL-13 antibody neutralises naturally occurring IL-13with a potency that is equal to or better than the potency of an IL-13antigen binding site formed by BAK502G9 VH domain (SEQ ID NO:15 in WO2005/007699) and the BAK502G9 VL domain (SEQ ID NO:16 in WO2005/007699).

Preferably, the IL-13 antibody is a monoclonal IL-13 antibody, morepreferably a human IL-13 monoclonal antibody.

A particularly preferred IL-13 antibody is one selected from thosedescribed in WO 2003/035847, WO 2003/086451, WO 2005/007699 or WO2005/081873.

For example, in one particularly preferred embodiment, the IL-13antibody is BAK278D6 HCDR1-3 and LCDR1-3 (SEQ ID NOS: 1-6 in WO2005/007699, respectively). A set of CDR's with the BAK278D6 set ofCDR's, BAK278D6 set of HCDR's or BAK278D6 LCDR's, or one or twosubstitutions therein, is said to be of the BAK278D6 lineage.

In a further particularly preferred embodiment, the IL-13 antibody isBAK502G9 HCDR1-3 and LCDR1-3 (SEQ ID NOS: 7-12 in WO 2005/007699,respectively).

In a yet further particularly preferred embodiment, the IL-13 antibodyis BAK1111D10 HCDR1-3 and LCDR1-3 (SEQ ID NOS: 91-96 in WO 2005/007699,respectively).

In a yet further particularly preferred embodiment, the IL-13 antibodyis BAK1167F2 HCDR1-3 and LCDR1-3 (SEQ ID NOS: 61-66 in WO 2005/007699,respectively).

In a yet further particularly preferred embodiment, the IL-13 antibodyis BAK1183H4 HCDR1-3 and LCDR1-3 (SEQ ID NOS: 97-102 in WO 2005/007699,respectively).

The relevant set of CDR's is provided within antibody framework regionsor other protein scaffolds, e.g. fibronectin or cytochrome B [Koide etal., J. Mol. Biol. 1998. 284, 1141-1151; Nygren et al., Current Opinionin Structural Biology, 1997. 7, 463-469]. Preferably antibody frameworkregions are employed, and where they are employed they are preferablygermline, more preferably the antibody framework region for the heavychain may be DP14 from the VH1 family. The preferred framework regionfor the light chain may be X3-3H. For the BAK502G9 set of CDR's it ispreferred that the antibody framework regions are for VH FR1-3, SEQ IDNOS: 27-29 in WO 2005/007699, respectively and for light chain FR1-3,SEQ ID NOS: 30-32 in WO 2005/007699, respectively. In a preferredembodiment, a VH domain is provided with the amino acid sequence of SEQID NO: 15 in WO 2005/007699, this being termed “BAK502G9 VH domain”. Ina further highly preferred embodiment, a VL domain is provided with theamino acid sequence of SEQ ID NO: 16 in WO 2005/007699, this beingtermed “BAK502G9 VL domain.

In a preferred embodiment, the IL-13 antibody cross reacts withcynomologous IL-13 and/or the IL-13 variant, Q130R.

Preferably, the IL-13 antibody is present within the pharmaceuticalcomposition in an amount of between 1 and 200 mg/ml, more preferably 50and 100 mg/ml, especially 50 mg/ml.

Preferably, the pharmaceutical composition is buffered to a pH of 5.2 to5.7, most preferably 5.5±0.1. The selection of such a pH conferssignificant stability to the pharmaceutical composition. Examples ofalternative buffers that control the pH in this range include succinate,gluconate, histidine, citrate, phosphate, glutaric, cacodylyte, sodiumhydrogen maleate, tris-(hydroxylmethyl)aminomethane (Tris),2-(N-morpholino)ethanesulphonic acid (MES), imidazole and other organicacid buffers.

Preferably, the buffer is acetate buffer, more preferably sodiumacetate.

Preferably, the acetate buffer is present within the pharmaceuticalcomposition in an amount of between 1 and 100 mM, more preferably 30 and70 mM, especially 50 mM.

It will be appreciated that references to “pharmaceutically acceptableexcipient” includes references to any excipient conventionally used inpharmaceutical compositions. Such excipients may typically include oneor more surfactant, inorganic or organic salt, stabilizer, diluent,solubilizer, reducing agent, antioxidant, chelating agent, preservativeand the like. Examples of a typical surfactant include: nonionicsurfactants (HLB 6 to 18) such as sorbitan fatty acid esters (e.g.sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate),glycerine fatty acid esters (e.g. glycerine monocaprylate, glycerinemonomyristate, glycerine monostearate), polyglycerine fatty acid esters(e.g. decaglyceryl monostearate, decaglyceryl distearate, decaglycerylmonolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g.polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate,polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fattyacid esters (e.g. polyoxyethylene sorbitol tetrastearate,polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerine fattyacid esters (e.g. polyoxyethylene glyceryl monostearate), polyethyleneglycol fatty acid esters (e.g. polyethylene glycol distearate),polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether),polyoxyethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylenealkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether),polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castoroil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswaxderivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylenelanolin derivatives (e.g. polyoxyethylene lanolin), and polyoxyethylenefatty acid amides (e.g. polyoxyethylene stearyl amide);

anionic surfactants such as C₁₀-C₁₈ alkyl sulfates salts (e.g. sodiumcetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate),polyoxyethylene C₁₀-C₁₈ alkyl ether sulfates salts with an average of 2to 4 moles of ethylene oxide (e.g. sodium polyoxyethylene laurylsulfate), and C₈-C₁₈ alkyl sulfosuccinate ester salts (e.g. sodiumlauryl sulfosuccinate ester); and natural surfactants such as lecithin,glycerophospholipid, sphingophospholipids (e.g. sphingomyelin), andsucrose esters of C₁₂-C₁₈ fatty acids.

Preferably, the surfactant is selected from polyoxyethylene sorbitanfatty acid esters. Particularly preferably the surfactant is Polysorbate20, 21, 40, 60, 65, 80, 81 and 85, most preferably Polysorbate 20 and80, especially Polysorbate 80.

Preferably, the surfactant is present within the pharmaceuticalcomposition in an amount of between 0.001 and 0.1% (w/w), morepreferably 0.005 and 0.05 (w/w), especially 0.01% (w/w).

Examples of a typical inorganic salt include: sodium chloride, potassiumchloride, calcium chloride, sodium phosphate, sodium sulphate, ammoniumsulphate, potassium phosphate and sodium bicarbonate or any othersodium, potassium or calcium salt. Preferably, the inorganic salt issodium chloride.

Preferably, the inorganic salt is present within the pharmaceuticalcomposition in an amount of between 10 and 200 mM, more preferably 60and 130 mM, especially 85 mM.

Examples of a reducing agent include N-acetylcysteine,N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine,thioglycerol, thiosorbitol, thioglycolic acid and a salt thereof, sodiumthiosulfate, glutathione, and a C1-C7 thioalkanoic acid.

Examples of an antioxidant include erythorbic acid,dibutylhydroxytoluene, butylhydroxyanisole, alpha-tocopherol, tocopherolacetate, L-ascorbic acid and a salt thereof, L-ascorbic acid palmitate,L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamylgallate and propyl gallate.

Examples of a chelating agent include disodiumethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodiummetaphosphate.

Examples of a stabiliser include creatinine, an amino acid selected fromhistidine, alanine, glutamic acid, glycine, leucine, phenylalanine,methionine, isoleucine, proline, aspartic acid, arginine, lysine andthreonine, a carbohydrate selected from sucrose, trehalose, sorbitol,xylitol and mannose, surfactants selected from polyethylene glycol (PEG;e.g. PEG3350 or PEG4000) or polyoxyethylene sorbitan fatty acid esters(e.g. Polysorbate 20 or Polysorbate 80), or any combination thereof.

In one preferred embodiment the stabiliser comprises a singlecarbohydrate as hereinbefore defined (e.g. trehalose).

In an alternatively preferred embodiment the stabilizer comprises anamino acid in combination with a carbohydrate (e.g. trehalose andalanine or trehalose, alanine and glycine).

In a further alternatively preferred embodiment the stabiliser comprisesan amino acid in combination with a carbohydrate and a surfactant (e.g.trehalose, alanine and PEG3350 or trehalose, proline and PEG3350 ortrehalose, alanine and Polysorbate 80 or trehalose, proline andPolysorbate 80 or trehalose, alanine, glycine and PEG3350 or trehalose,alanine, glycine and Polysorbate 80).

In a yet further alternatively preferred embodiment the stabilisercomprises an amino acid in combination with a surfactant (e.g. alanineand PEG3350 or alanine, glycine and PEG3350).

In a yet further alternatively preferred embodiment the stabilisercomprises a carbohydrate in combination with a surfactant (e.g.trehalose and PEG3350 or trehalose and Polysorbate 80).

Examples of a preservative include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), benzethonium chloride, aromatic alcohols such asphenol, butyl and benzyl alcohol, alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, andm-cresol.

In a preferred embodiment of the invention, the pharmaceuticalcomposition comprises an IL-13 antibody, a surfactant and an inorganicsalt buffered to a pH of 5.5±0.1 with acetate buffer.

In a further preferred embodiment of the invention, the pharmaceuticalcomposition comprises an IL-13 antibody, sodium chloride and Polysorbate80 buffered to a pH of 5.5±0.1 with sodium acetate buffer.

In a yet further preferred embodiment of the invention, thepharmaceutical composition comprises 50 mg/ml of an IL-13 antibody, 85mM sodium chloride and 0.01% (w/w) Polysorbate 80 buffered to a pH of5.5±0.1 with 50 mM sodium acetate buffer.

According to a second aspect of the invention there is provided aprocess for purifying an IL-13 antibody which comprises one or morechromatographic separation steps wherein each of said separation stepscomprises elution with an elution buffer comprising one or morepharmaceutically acceptable excipients buffered to a pH of 3.5-7.0 withacetate buffer.

Preferably, the one or more chromatographic separation steps areselected from affinity chromatography (e.g. Protein A or Protein Gaffinity chromatography), ion exchange chromatography (e.g. cation andanion exchange chromatography), hydrophobic interaction chromatography(e.g. phenyl chromatography), hydroxyapatite chromatography, sizeexclusion chromatography, immobilised metal affinity chromatography,hydrophilic interaction chromatography, thiophilic adsorptionchromatography, euglobulin adsorption chromatography, dye ligandchromatography or immobilised boronate chromatography. Most preferably,chromatographic separation is performed by Protein A affinitychromatography followed by cation exchange chromatography (e.g. using anSP-sepharose matrix) followed by anion exchange chromatography (e.g.using a Q-sepharose matrix).

Preferably, the one or more pharmaceutically acceptable excipientscomprises an inorganic salt such as sodium chloride.

Preferably, the inorganic salt is present within the elution buffer inan amount of between 10 and 200 mM, more preferably 60 and 130 mM,especially 85 mM.

Examples of alternative buffers that control the pH in the range of3.5-7.0 include succinate, gluconate, histidine, citrate, phosphate andother organic acid buffers.

Preferably, the buffer is acetate buffer, more preferably sodiumacetate.

Preferably, the acetate buffer is present within the elution buffer inan amount of between 1 and 100 mM, more preferably 30 and 70 mM,especially 50 mM.

Most preferably, the elution buffer comprises 50 mM sodium acetate and85 mM sodium chloride buffered to pH 5.5±0.1.

A nucleic acid encoding any IL-13 antibody of the invention (e.g. CDR orset of CDR's or VH domain or VL domain or antibody antigen-binding siteor antibody molecule, e.g. scFv or IgG4 as provided), may be expressedby culturing under appropriate conditions recombinant host cellscontaining said nucleic acid. Following production by expression a VH orVL domain, or specific binding member may be isolated and/or purifiedusing any suitable technique, then used as appropriate.

Specific binding members, VH and/or VL domains, and encoding nucleicacid molecules and vectors may be provided isolated and/or purified,e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or gene origin other than the sequence encoding apolypeptide with the required function. Nucleic acid may comprise DNA orRNA and may be wholly or partially synthetic.

Reference to a nucleotide sequence as set out herein encompasses a DNAmolecule with the specified sequence, and encompasses an RNA moleculewith the specified sequence in which U is substituted for T, unlesscontext requires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, yeast and baculovirus systemsand transgenic plants and animals.

Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 ratmyeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Preferably, said mammalian cell line is a myeloma cell culture such ase.g. NS0 cells [Galfre and Milstein Methods Enzymology, 1981. 73, 3].Myeloma cells are plasmacytoma cells, i.e. cells of lymphoid celllineage. An exemplary NS0 cell line is e.g. cell line ECACC No.85110503, freely available from the European Collection of Cell Cultures(ECACC), Centre for Applied Microbiology & Research, Salisbury,Wiltshire, SP4 0JG, United Kingdom. NS0 have been found able to giverise to extremely high product yields, in particular if used forproduction of recombinant antibodies.

An alternatively preferred mammalian cell line is Chinese hamster ovary(CHO) cells. These may be dihydrofolate reductase (dhfr) deficient andso dependent on thymidine and hypoxanthine for growth [PNAS, 1990. 77,4216-4220]. The parental dhfr CHO cell line is transfected with theantibody gene and dhfr gene which enables selection of CHO celltransformants of dhfr positive phenotype.

Selection is carried out by culturing the colonies on media devoid ofthymidine and hypoxanthine, the absence of which prevents untransformedcells from growing and transformed cells from resalvaging the folatepathway and thereby bypassing the selection system. These transformantsusually express low levels of the product gene by virtue ofco-integration of both transfected genes. The expression levels of theantibody gene may be increased by amplification using methotrexate(MTX). This drug is a direct inhibitor of the dhfr enzyme and allowsisolation of resistant colonies which amplify their dhfr gene copynumber sufficiently to survive under these conditions. Since the dhfrand antibody genes are more closely linked in the originaltransformants, there is usually concomitant amplification, and thereforeincreased expression of the desired antibody gene.

Another selection system for use with CHO or myeloma cells is theglutamine synthetase (GS) amplification system described in WO 87/04462.This system involves the transfection of a cell with a gene encoding theGS enzyme and the desired antibody gene. Cells are then selected whichgrow in glutamine free medium. These selected clones are then subjectedto inhibition of the GS enzyme using methionine sulphoximine (MSX). Thecells, in order to survive, will amplify the GS gene with concomitantamplification of the gene encoding the antibody.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Pluckthun, A. Bio/Technology 1991. 9, 545-551. Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member for example[Chadd, H. E. and Chamow, S. M., Current Opinion in Biotechnology 2001.12, 188-194; Andersen, D. C. and Krummen, L. Current Opinion inBiotechnology 2002. 13, 117; Larrick, J. W. and Thomas, D. W. Currentopinion in Biotechnology 2001. 12, 411-418].

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.'phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrookand Russell, 2001, Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds. , John Wiley & Sons, 1988,Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, Ausubel et al. eds., John Wiley& Sons, 4th edition 1999. The disclosures of Sambrook et al. and Ausubelet al. (both) are incorporated herein by reference.

Introduction of a nucleic acid into a host cell may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay be incorporated into the host cell or into an artificial chromosome[Csonka, E. et al., Journal of Cell Science, 200. 113, 3207-3216;Vanderbyl, S. et al., Molecular Therapy, 2002. 5 (5), 10]. Incorporationmay be either by random or targeted integration of one or more copies atsingle or multiple loci. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation and infectionusing bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e. g. chromosome) of the host cell.

Integration may be promoted by inclusion of sequences which promoterecombination with the genome, in accordance with standard techniques.

According to a third aspect of the invention there is provided a use ofa pharmaceutical antibody composition as defined herein in themanufacture of a medicament for the treatment of an IL-13 relateddisorder.

Preferably, the IL-13 related disorder is selected from asthma, atopicdermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonarydisease, scleroderma, inflammatory bowel disease and Hodgkin's lymphoma.The composition of the invention may also be used in the treatment oftumours and viral infections as IL-13 antibodies will inhibit IL-13mediated immunosuppression. Most preferably, the IL-13 related disorderis asthma.

The invention further provides a method of treatment or prophylaxis ofan IL-13 related disorder which comprises administering to the sufferera therapeutically effective amount of a pharmaceutical antibodycomposition as defined herein.

The invention further provides a pharmaceutical antibody composition asdefined herein for use in the treatment of an IL-13 related disorder.

The pharmaceutical composition of the invention may be a liquidformulation or a lyophilized formulation which is reconstituted beforeuse. As excipients for a lyophilized formulation, for example, sugaralcohols or saccharides (e.g. mannitol or glucose) may be used. In thecase of a liquid formulation, the pharmaceutical composition of theinvention is usually provided in the form of containers with definedvolume, including sealed and sterilized plastic or glass vials, ampoulesand syringes, as well as in the form of large volume containers likebottles. Preferably, the composition of the invention is a liquidformulation.

The pharmaceutical composition of the invention may be administeredorally, by injection (for example, subcutaneously, intravenously,intraperitoneal or intramuscularly), by inhalation, or topically (forexample intraocular, intranasal, rectal, into wounds, on skin). Theroute of administration can be determined by the physicochemicalcharacteristics of the treatment, by special considerations for thedisease or by the requirement to optimise efficacy or to minimiseside-effects.

Preferably, the composition of the invention is administered bysubcutaneous injection. It is envisaged that treatment will not berestricted to use in the clinic. Therefore, subcutaneous injection usinga needle free device may also be preferred.

In accordance with the invention, compositions provided may beadministered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors. Appropriate doses of antibody are well known inthe art; [Ledermann, J. A. et al., Int. J. Cancer, 1999. 47, 659-664;Bagshawe, K. D. et al., Antibody, Immunoconjugates andRadiopharmaceuticals, 1991. 4, 915-922].

The precise dose will depend upon a number of factors, including thesize and location of the area to be treated, the precise nature of theantibody (e. g. whole antibody, fragment or diabody), and the nature ofany detectable label or other molecule attached to the antibody. Atypical antibody dose will be in the range 100 pg to 10 g for systemicapplications, and 1 μg to 100 mg for topical applications.

Typically, the antibody will be a whole antibody, preferably the IgG4isotype. A dose for a single treatment of an adult patient, may beproportionally adjusted for children and infants, and also adjusted forother antibody formats in proportion to molecular weight. Treatments maybe repeated at daily, twice-weekly, weekly or monthly intervals, at thediscretion of the physician. In preferred embodiments of the presentinvention, treatment is periodic, and the period between administrationsis about two weeks or more, preferably about three weeks or more, morepreferably about four weeks or more, or about once a month.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of SDS-PAGE analysis of samples at Day 1 of a28 day stability assessment of the composition of the invention.

FIG. 2 shows the results of SDS-PAGE analysis of samples at Day 21 of a28 day stability assessment of the composition of the invention.

FIG. 3 shows the results of GP-HPLC analysis of samples at Day 1 of a 28day stability assessment of the composition of the invention.

FIG. 4 shows an amalgam of results obtained from GP-HPLC analysis ofsamples during a 28 day stability assessment of the composition of theinvention at 2-8° C.

FIG. 5 shows an amalgam of results obtained from GP-HPLC analysis ofsamples during a 28 day stability assessment of the composition of theinvention at 25° C.

FIG. 6 shows an amalgam of results obtained from GP-HPLC analysis ofsamples during a 28 day stability assessment of the composition whenneutralised with differing buffers.

FIG. 7 shows the results of IEF analysis of samples at Day 28 of a 28day stability assessment of the composition of the invention.

FIG. 8 shows the results of gel filtration HPLC analysis of a 12 monthstability assessment of different formulations stored at −70° C.

FIG. 9 shows the results of gel filtration HPLC analysis of a 12 monthstability assessment of different formulations stored at +5° C.

FIG. 10 shows the results of gel filtration HPLC analysis of a 12 monthstability assessment of different formulations stored at +25° C.

FIG. 11 shows the results of gel filtration HPLC analysis of an 8 weekstability assessment of different formulations stored at +37° C.

FIG. 12 shows the results of gel filtration HPLC analysis of a 5 daystability assessment of different formulations stored at +45° C.

FIG. 13 shows the results of reduced SDS-PAGE analysis at 0, 6 and 12months during a 12 month stability assessment of different formulationswhen stored at −70° C.

FIG. 14 shows the percentage abundance of BAK502G9 heavy and lightchains following reduced SDS-PAGE analysis in FIG. 13 when stored at−70° C.

FIG. 15 shows the results of reduced SDS-PAGE analysis at 0, 6 and 12months during a 12 month stability assessment of different formulationswhen stored at +5° C.

FIG. 16 shows the percentage abundance of BAK502G9 heavy and lightchains following reduced SDS-PAGE analysis in FIG. 15 when stored at +5°C.

FIG. 17 shows the results of reduced SDS-PAGE analysis at 0, 6 and 12months during a 12 month stability assessment of different formulationswhen stored at +25° C.

FIG. 18 shows the percentage abundance of BAK502G9 heavy and lightchains following reduced SDS-PAGE analysis in FIG. 17 when stored at+25° C.

FIG. 19 shows the results of reduced SDS-PAGE analysis at 0 and 8 weeksduring an 8 week stability assessment of different formulations whenstored at +37° C.

FIG. 20 shows the percentage abundance of BAK502G9 heavy and lightchains following reduced SDS-PAGE analysis in FIG. 19 when stored at+37° C.

FIG. 21 shows the results of reduced SDS-PAGE analysis at 0 and 5 daysduring a 5 day stability assessment of different formulations whenstored at +45° C.

FIG. 22 shows the percentage abundance of BAK502G9 heavy and lightchains following reduced SDS-PAGE analysis in FIG. 21 when stored at+45° C.

FIG. 23 shows the results of non-reduced SDS-PAGE analysis at 0, 6 and12 months during a 12 month stability assessment of differentformulations when stored at −70° C.

FIG. 24 shows the percentage abundance of intact BAK502G9 monomerfollowing non-reduced SDS-PAGE analysis in FIG. 23 when stored at −70°C.

FIG. 25 shows the results of non-reduced SDS-PAGE analysis at 0, 6 and12 months during a 12 month stability assessment of differentformulations when stored at +5° C.

FIG. 26 shows the percentage abundance of intact BAK502G9 monomerfollowing non-reduced SDS-PAGE analysis in FIG. 25 when stored at +5° C.

FIG. 27 shows the results of non-reduced SDS-PAGE analysis at 0, 6 and12 months during a 12 month stability assessment of differentformulations when stored at +25° C.

FIG. 28 shows the percentage abundance of intact BAK502G9 monomerfollowing non-reduced SDS-PAGE analysis in FIG. 27 when stored at +25°C.

FIG. 29 shows the results of non-reduced SDS-PAGE analysis at 0 and 8weeks during an 8 week stability assessment of different formulationswhen stored at +37° C.

FIG. 30 shows the percentage abundance of intact BAK502G9 monomerfollowing non-reduced SDS-PAGE analysis in FIG. 29 when stored at +37°C.

FIG. 31 shows the results of non-reduced SDS-PAGE analysis at 0 and 5days during a 5 day stability assessment of different formulations whenstored at +45° C.

FIG. 32 shows the percentage abundance of intact BAK502G9 monomerfollowing non-reduced SDS-PAGE analysis in FIG. 31 when stored at +45°C.

The present invention will now be illustrated, merely by way of example,with reference to the following methods and examples.

EXAMPLE 1 Expression of BAK502G9

BAK502G9 was expressed in a GS NSO cell line in an analogous manner tothe procedures described in WO 87/04462 and WO 2004/076485 and yieldedculture supernatant containing 598 mg/1 BAK502G9 antibody.

EXAMPLE 2 Purification of BAK502G9

(a) rmp Protein A Sepharose Purification

The column used for the rmp Protein A Sepharose fast flow chromatographystep was 2.6 cm diameter, packed in 0.9% ^(w)/_(v) sodium chloride, to abed height of 14.5 cm giving a column volume of 77 ml. Resin was sourcedfrom GE Healthcare/Amersham Biosciences 17-5138. Chromatography wasperformed using an Amersham Biosciences P-50 pump, UV1 detector and flowcell. The column was cleaned with 6M guanidine hydrochloride prior touse.

The rmp Protein A Sepharose fast flow column was subsequentlyequilibrated with 350 ml phosphate buffered saline pH 7.2, followingwater wash and cleaning. 2.6 l culture supernatant was loaded directlyonto the column at 200 cm/hour and room temperature.

The column was then washed with 567 ml phosphate buffered saline pH 7.2followed by 568 ml 50 mM sodium acetate pH 5.55. BAK502G9 antibody waseluted from the column by washing with 380ml 50 mM sodium acetate pH3.75. Post elution the column was washed with 50 mM acetic acid pH3.0.The elution peak was collected between 2% maximum UV deflection on theup and down slope of the peak. 1.5 g BAK502G9 antibody was recovered in217 ml.

(b) Low pH Viral Inactivation

The rmp Protein A Sepharose fast flow eluate was adjusted to pH 3.70with 173 ml 100 mM acetic acid. The adjusted eluate was then held for 60minutes for viral inactivation. After this time the adjusted eluate wasneutralised with 437ml 50 mM sodium hydroxide to pH 5.50 and 0.22 μmfiltered using a Millipore stericup (product number SCGPU11RE). 1.4 gBAK502G9 antibody was recovered in 823 ml.

(c) SP Sepharose Purification

The column used for the SP Sepharose fast flow chromatography step was1.6 cm diameter, packed in phosphate buffered saline pH 7.2, to a bedheight of 15.5 cm giving a column volume of 31 ml. Resin was sourcedfrom GE Healthcare/Amersham Biosciences 17-0729. Chromatography wasperformed using an Amersham Biosciences P-50 pump, UV1 detector and flowcell. The column was cleaned with 0.5 M sodium hydroxide prior to use.

The SP Sepharose fast flow column was subsequently equilibrated with 145ml 50 mM sodium acetate pH 5.50, following water wash and cleaning.

400 ml BAK502G9 antibody as neutralised rmp Protein A eluate was loadeddirectly onto the column at 200 cm/hour and room temperature.

The column was then washed with 302 ml 50 mM sodium acetate+30 mM sodiumchloride pH 5.50.

BAK502G9 antibody was eluted from the column by washing with 150ml 50 mMsodium acetate+85 mM sodium chloride pH 5.50. Post elution the columnwas washed with 50 mM sodium acetate+2M sodium chloride pH 5.50.

The elution peak was collected between 2% maximum UV deflection on theup slope and 30% on the down slope of the peak. Eluate was 0.22 μmfiltered using a Millipore steriflip (product number SCGPOO525). 0.67 gBAK502G9 antibody was recovered in 54 ml.

This process step was repeated with the remaining 392 ml BAK502G9antibody as neutralised rmp Protein A eluate. A further 0.67 g BAK502G9antibody was recovered in 54 ml. The two filtered SP Sepharose eluateswere pooled prior to the next step.

(d) Q Sepharose Fast Flow Chromatography

The column used for the Q Sepharose fast flow chromatography step was1.6 cm diameter, packed in phosphate buffered saline pH 7.2, to a bedheight of 13.3 cm giving a column volume of 27 ml. Resin was sourcedfrom GE Healthcare/Amersham Biosciences 17-0510. Chromatography wasperformed using an Amersham Biosciences P-50 pump, UV1 detector and flowcell. The column was cleaned with 0.5M sodium hydroxide prior to use.

The Q Sepharose fast flow column was subsequently equilibrated with 138ml 50 mM sodium acetate+85 mM sodium chloride pH 5.50, following waterwash and cleaning.

44 ml BAK502G9 antibody as SP Sepharose eluate was loaded directly ontothe column at 200 cm/hour and room temperature.

Isocratic elution of the BAK502G9 antibody was undertaken by washing thecolumn with 124 ml 50 mM sodium acetate+85 mM sodium chloride pH 5.50.Post elution the column was washed with 50 mM sodium acetate+2M sodiumchloride pH 5.50.

The elution peak was collected between 2% maximum UV deflection on theup slope and 2% on the down slope of the peak. Eluate was 0.22 μmfiltered using a Millipore stericup (product number SCGPU01RE). 0.52 gBAK502G9 antibody was recovered in 88 ml. This process step was repeatedwith the remaining 44 ml BAK502G9 antibody as SP Sepharose eluate. Afurther 0.54 g BAK502G9 antibody was recovered in 51 ml. The twofiltered Q Sepharose eluates were then pooled.

(e) Concentration

The product was obtained in 50 mM sodium acetate+85 mM sodium chloridepH 5.50 and did not require diafiltration.

95.31 g BAK502G9 antibody as Q Sepharose eluate in 17.5 l wasconcentrated to 1.5 l. A Millipore Pellicon 2 TFF system and a 0.1 M² 30KDa membrane (Millipore P2B030A01) were used to carry out theconcentration. The BAK502G9 antibody was then recovered from theequipment, the equipment was then buffer flushed with 50 mM sodiumacetate+85 mM sodium chloride pH 5.50. The concentrated antibody wasthen combined with the buffer flush. 1.67 ml 10% ^(w)/_(v) Polysorbate80 was added to the pool to give a final Polysorbate 80 concentration of0.01% ^(w)/_(v). The pool was then 0.22 μm filtered. 91.6 g BAK502G9antibody was recovered in 1.67 l.

(f) Materials Used

The chemicals used to prepare the above buffers were as follows:

-   Guanidine hydrochloride. Sigma Aldrich. G4505-   Di sodium hydrogen orthophosphate. VWR. 1038349-   Sodium di hydrogen orthophosphate. VWR. 102454R-   Sodium acetate 3-hydrate. VWR. 102354X.-   Sodium chloride. VWR. 10241AP.-   Acetic acid. VWR. 10001CU.-   Polysorbate 80. J. T. Baker. 7394.

EXAMPLE 3 Day Stability Analysis

Methodology

rmp Protein A chromatography was performed as set out in Table 1 below:

TABLE 1 Protein A Chromatography Parameters Matrix: rmp Protein ASepharose Bed height: 14.2 cm Column diameter 5.0 cm Column Volume 278ml Linear flow rate: 150 cm/hr Equilibration buffer & Phosphate bufferedsaline, pH 7.2, CV's: 5 CV's Load material: Clarified culture harvest.Load capacity (mg IgG/ml 16.6 mg/ml matrix matrix): Wash 1 buffer andCV's: Phosphate buffered saline, pH 7.2, 7.5 CV's Wash 2 buffer andCV's: 50 mM sodium acetate, pH 5.50 ± 0.10, 7.5 CV's Elution buffer: 50mM sodium acetate, pH 3.75 ± 0.10, 1.6 CV's Strip/Clean buffer and 50 mMsodium acetate, pH 3.0 ± 0.10, CV's: 2 CVs.

1.6 CV of elution buffer was required to elute the peak and for theabsorbance to return to <2% full scale deflection (AuFS). After this thebuffer was changed to strip buffer.

Low pH virus inactivation was then performed using the Protein A eluateas set out in Table 2 below:

TABLE 2 Low pH virus inactivation Start material: Protein A Eluate pHreduction: To pH 3.70 Buffer for reduction of pH: 100 mM acetic acidRate of addition: 8.6 ml/min (19.0 ml/min/l eluate) Hold time at low pH:60 minutes post pH adjustment Neutralisation post virus To pH 5.48 bythe gradual addition of inactivation: 100 mM sodium hydroxide. Rate ofaddition: 17.8 ml/min (39.3 ml/min/l eluate - adjusted for sampling)Filtration post Millipak 20 neutralisation:

All samples were stored in 200 ml Hyclone BioProcess containers whichare made of polyethylene with C-flex inlet and outlet tubing attached.

In addition, a 50 ml sample of virus inactivated Protein A eluate wasneutralised with the original buffer (50 mM sodium hydroxide) and storedin a bioprocess container at 2 to 8° C. for twenty-eight days beforeanalysis.

All samples were either stored in a class 100,000 cold room (2 to 8° C.,monitored with chart recorder and alarm) or in a thermostaticallycontrolled incubator set to 25° C., as appropriate.

There were five time points and two storage temperatures (2 to 8° C. and25° C.) i.e. ten separate sampling points.

The filtered neutralised Protein A eluate was split into ten lots; fivelots being stored at each of the temperatures. A further container ofunfiltered, neutralised Protein A eluate was stored at 2 to 8° C.

To fill each bioprocess container, the neutralised Protein A eluate waspumped through 0.2 μm Millipak filter into a 200 ml bioprocesscontainer. Each bioprocess container was filled with approximately 100ml to mimic the ratio of product to surface contact envisaged for thefinal 2,000 l scale production.

One bioprocess container was used for sampling for each temperature andeach time-point and care was taken not to leave material in the tubingduring storage.

One bioprocess container from each temperature was removed on days 1, 3,15, 21 and 28. The contents of each bioprocess container were allowed toreach room temperature prior to sampling.

Samples were taken into Bijoux containers or 50 ml Falcon tubes. Thefirst few mls of sample were discarded as they may have had some contactwith the tubing during storage.

Post sampling the containers containing the remaining material werefrozen at −70° C.

The following analysis was carried out on the day of each time point andfor each storage temperature to determine comparability of these samplesto each other and the reference standard. A fresh vial of BAK502G9standard was used at each time point for comparison to the stabilitystudy material. The reference was thawed at room temperature from −70°C. storage. It is known that multimer levels can be elevated shortlyfollowing thawing of this antibody, so the GP-HPLC analysis was carriedout last.

(A) Turbidity Analysis

Turbidity was assessed as a measure of protein degradation over time andtherefore serves as a useful stability assessment of the composition ofthe invention.

Turbidity was measured by taking the average absorbance of the samplebetween A340 and 360nm (Eckhardt et al. 1993). This assay was carriedout without further filtration.

Data obtained from the rmp Protein A chromatography are summarised inTable 3 and the chromatogram is shown in FIG. 1.

TABLE 3 rmp Protein A chromatography data Step Details Result Columnvolume 278 ml Total load volume 3204 ml Total product loaded 4620 mgEluate volume 453 ml (1.6 CV) Concentration of eluate 8.42 mg/ml pH onelution 4.40 Volume of 100 mM 665 ml (2.4 CV) acetic acid added Volumeof 100 mM 838 ml (3.0 CV; sodium hydroxide added 3.2 CV adjusted*)Volume pre in process 1900 ml* (6.8 CV) filtration Concentration post in2.28 mg/ml process filtration Total product post in 4327 mg processfiltration Percentage recovery 93.7% post in process filtration *50 mlremoved from virus-inactivated eluate for adjustment with 50 mM sodiumhydroxide. Final volume is post sampling.

rmp Protein A chromatography performed as expected generating volumes ofbuffers comparable to that seen at large scale. The neutralised eluatewas visibly more turbid than is usually seen with eluate neutralisedwith 50 mM sodium hydroxide. During filtration in to bioprocesscontainers, precipitation began to be observed, this occurred within onehour of neutralisation. Turbidity was not assayed until after this time.Recovery was within the expected range post filtration.

All eluate samples were assayed for turbidity on day one. Filteredeluates stored at both temperatures were assayed for turbidity on days:one, three, fifteen, twenty-one and twenty-eight. In addition eluatesstored unfiltered (adjusted with either 50 mM sodium hydroxide or 100 mMsodium hydroxide) were assayed for turbidity both pre and postfiltration at day twenty-eight. The results of the turbidity analysisare shown in Table 4 where it can be seen that although there was anoticeable increase in turbidity of the day twenty-eight sample storedat 25° C., the samples were generally stable at 25° C. for at least 21days.

TABLE 4 Turbidity measurements of process and stability study samplesSample Ave Turbidity Category Unadjusted eluate 0.0640 Opalescent Virusinactivated 0.0247 Slightly Opalescent Neutralised pre filtration 0.2856Cloudy Filtered eluate 0.0093 Clear 2 to 8° C. 25° C. Day TurbidityCategory Turbidity Category Day one. 0.0093 Clear 0.0132 Clear Daythree. 0.0115 Clear 0.0176 Slightly Opalescent Day fifteen. 0.0208Slightly 0.0203 Slightly Opalescent Opalescent Day twenty-one. 0.0120Clear 0.0202 Slightly Opalescent Day twenty-eight 0.0140 Clear 0.0412Opalescent

(B) Protein Concentration Analysis

IgG concentration was measured and calculated using absorbance at 280 nmand an extinction coefficient of E_(1 cm) ^(0.1%)=1.723.

The eluates that were filtered at the time of neutralisation were notre-filtered prior to absorbance at 280 nm being measured for calculatingprotein concentrations. As the turbidities were all low it is believedthis would not have affected the results. Absorbance at 280 nm wasconsistent between storage temperatures and time points which is shownin Table 5.

TABLE 5 Protein concentrations of eluate samples Sample: Proteinconcentration Filtered neutralised Protein A eluate (mg/ml) neutralizedwith 100 mM sodium hydroxide Temperature Day: 2 to 8° C. 25° C. One.2.28 2.28 Three. 2.29 2.29 Fifteen. 2.28 2.29 Twenty-one. 2.29 2.28Twenty-eight. 2.28 2.26 Twenty-eight. Re-filtered. 2.29 —

(C) SDS-PAGE Analysis

Reduced and non-reduced SDS PAGE was run using 4 to 12% Bis-Tris NuPAGEgels. Gels were run using MES running buffer and stained with Pierce gelcode blue.

SDS PAGE analysis demonstrated variations in levels of half antibody,but there is no apparent trend and it is likely this is within thevariation of the assay (see FIGS. 1 and 2 and Tables 6 and 7). At daytwenty-one, some additional minor bands were seen in the 25° C. sample.These bands were not seen at day twenty-eight. Therefore these may be atthe limits of detection of the assay. These bands may correspond withthe small additional peaks seen on GP-HPLC (see Example 3D).

Samples were not run at the end of the study, as it would not bepossible to distinguish between differences that were present in theoriginal sample or were a result of the longer storage. A freshly thawedvial of reference standard was run on each gel. This was comparablebetween the different time points indicating that analysis had performedas expected. It is also notable that the extra bands seen in the daytwenty-one, 25° C. sample, were not seen in the reference standard or 2to 8° C. sample, run on the same gel. For clarity, all bands in FIG. 2are indicated with a circle.

TABLE 6 SDS PAGE densitometry results, reduced samples Percentage HeavyLight Half Day Sample Chain Chain antibody Others One. Reference. 61.137.2 1.7 0.0 2 to 8° C. 61.3 37.4 1.2 0.0 25° C. 61.5 37.5 1.0 0.0Three. Reference. 65.7 32.4 1.9 0.0 2 to 8° C. 64.4 33.4 2.1 0.0 25° C.66.0 32.8 1.2 0.0 Fifteen. Reference. 62.7 35.9 1.3 0.0 2 to 8° C. 62.337.3 0.4 0.0 25° C. 62.4 39.9 0.7 0.0 Twenty- Reference. 58.2 39.5 2.30.0 one. 2 to 8° C. 57.8 39.9 2.2 0.1 25° C. 55.9 40.0 2.5 1.6 Twenty-Reference. 64.4 33.7 1.9 0.0 eight. 2 to 8° C. 66.1 32.5 1.4 0.0 25° C.61.2 35.2 1.5 0.0

TABLE 7 SDS PAGE densitometry results, non - reduced samples PercentageDay Sample Whole antibody Half antibody Other One. Reference. 83.2 16.80.0 2 to 8° C. 82.6 17.4 0.0 25° C. 83.7 16.3 0.0 Three. Reference. 89.810.2 0.0 2 to 8° C. 87.1 12.9 0.0 25° C. 87.4 12.6 0.0 Fifteen.Reference. 86.0 14.0 0.0 2 to 8° C. 84.6 15.4 0.0 25° C. 86.8 13.2 0.0Twenty-one. Reference. 82.5 17.4 0.0 2 to 8° C. 81.6 17.9 0.5 25° C.82.5 15.7 1.8 Twenty- Reference. 87.1 12.9 0.0 eight. 2 to 8° C. 85.014.8 0.0 25° C. 87.9 11.3 0.0

(D) GP-HPLC Analysis

Samples were analysed using a TSK GS3000SW size exclusion column with200 mM sodium phosphate and 0.05% sodium azide pH 7.0 as the mobilephase and detection at 280 nm.

All fractions from Protein A chromatography were assayed on day 1.Eluates stored at both temperatures were then assayed on days: one,three, fifteen, twenty-one and twenty-eight. Reference samples were alsorun at all time points. Eluates separately neutralised with either 100mM sodium hydroxide or 50 mM sodium hydroxide on day one, but notfiltered until day twenty-eight were assayed on day twenty-eight byGP-HPLC.

Chromatograms from GP-HPLC analysis day one are shown in FIG. 3. Theseare as expected the final eluate being >95% monomer, which meets thefinal specification for BAK502G9.

GP-HPLC analysis of samples throughout the course of the study revealeda slight increase in truncated (in particular a small peak elutingbetween 9.9 and 10.3 minutes) BAK502G9 at 25° C. (Table 8 and FIGS. 4and 5). Levels of monomer were consistently between 97.0 and 98.2% overthe whole study.

Eluates neutralised with 50 mM sodium hydroxide had higher levels ofmonomer (99.2% monomer) than eluate neutralised with 100 mM sodiumhydroxide (97.4% monomer), both being higher than the required level of95% monomer for final product (Table 9 and FIG. 6).

The results obtained from the automatic integration were found to beinaccurate, therefore, the GP-HPLC chromatograms were manuallyre-integrated.

TABLE 8 GP-HPLC of eluates stored at different temperatures 2 to 8° C.(percentage) Trun- 25° C. (percentage) Day Multimer Monomer cateMultimer Monomer Truncate 1 1.4 98.2 0.4 1.4 98.0 0.7 3 1.7 97.5 0.8 1.098.3 0.7 15 1.4 98.0 0.6 1.0 98.0 1.0 21 1.2 98.1 0.6 1.3 97.4 1.3 281.2 98.2 0.7 1.5 97.0 1.5

TABLE 9 GP-HPLC of eluates neutralised with either 100 mM or 50 mMsodium hydroxide Multimer Monomer Truncate Fraction (%) (%) (%) Eluateneutralised with 100 mM 1.4 97.4 1.2 sodium hydroxide, filtered andassayed day 28 Eluate neutralised with 50 mM 0.3 99.2 0.5 sodiumhydroxide, filtered and assayed day 28 Filtered, eluate neutralised with1.4 98.2 0.4 100 mM sodium hydroxide, assayed day 28

(E): IEF Analysis

Samples were analysed using Invitrogen pH 3 to 10 IEF gels, usingInvitrogen buffers.

IEF analysis of BAK502G9 showed a consistent appearance throughout thisstudy (FIG. 7). Three major bands and two minor bands betweenapproximate pls of 7.1 and 6.4 are seen.

(F): Endotoxin Analysis

Samples taken on day twenty-eight from both storage temperatures wereassayed for endotoxin levels using a LAL assay.

Endotoxin levels in samples stored at both temperatures were low. Thisdemonstrates there has been no contamination by gram-negative bacteriaover the course of the study.

Eluate stored at 2 to 8° C.=0.87 EU/mg.

Eluate stored at 25° C.<0.44 EU/mg.

Summary of 28 Day Stability Analysis Results

BAK502G9 stored for up to fifteen days at 2 to 8° C. or 25° C. isequivalent in all the assays carried out in Example 3. After twenty-onedays some minor differences are observed by SDS PAGE (Example 3C) and GPHPLC (Example 3D) analysis, but the product remains comparable up totwenty-eight days.

EXAMPLE 4 Month Stability Analysis

The 12 month stability analysis was performed in an analogous manner tothat described for the 28 day stability analysis of Example 3.

The study was designed to investigate the stability of differentconcentrations of BAK502G9 when stored at different temperatures (e.g.−70, +5, +25, +37 and +45° C.). The differing formulations used in thisanalysis are set out in Table 10 below:

TABLE 10 Composition of formulations used in 12 month stability analysisNominal concentration Formulation (mg/ml) Composition Control 10 50 mMsodium acetate/85 mM sodium (CF) chloride pH 5.5 Test 1A 50 50 mM sodiumacetate/85 mM sodium (TF1A) chloride/0.01% Polysorbate 80 pH 5.5 Test 1B100 50 mM sodium acetate/85 mM sodium (TF1B) chloride/0.01% Polysorbate80 pH 5.5 Test 1C 150 50 mM sodium acetate/85 mM sodium (TF1C)chloride/0.01% Polysorbate 80 pH 5.5

Analysis of samples were taken at varying timepoints, for example, theformulations stored at −70, +5, +25° C. were measured at 0, 3, 6, 9 and12 months, the formulations stored at +37° C. were measured at 0, 1, 2,4 and 8 weeks and the formulations stored at +45° C. were measured at 0,1, 2 and 5 days.

(A) pH Analysis

pH was measured using a PHM220 pH meter (Radiometer Analytical) fittedwith a small volume pH electrode (BDH) and the results of pH measurementof formulations CF, TF1A, TF1B and TF1C at each temperature are shown inTables 11-15 below:

TABLE 11 pH after storage at −70° C. pH Time (months) CF TF1A TF1B TF1C0 5.67 5.52 5.51 5.54 3 5.47 5.48 5.48 NT 6 5.50 5.50 5.48 NT 9 5.565.55 5.57 5.58 12 5.46 5.47 5.46 5.46

TABLE 12 pH after storage at +5° C. pH Time (months) CF TF1A TF1B TF1C 05.67 5.52 5.51 5.54 3 5.48 5.49 5.49 NT 6 5.49 5.49 5.49 NT 9 5.56 5.565.55 5.56 12 5.47 5.47 5.46 5.47

TABLE 13 pH after storage at +25° C. pH Time (months) CF TF1A TF1B TF1C0 5.67 5.52 5.51 5.54 3 5.49 5.50 5.51 NT 6 5.49 5.50 5.48 NT 9 5.535.53 5.55 5.58 12 5.49 5.49 5.48 5.48

TABLE 14 pH after storage at +37° C. pH Time (weeks) CF TF1A TF1B TF1C 05.50 5.52 5.52 5.52 1 5.45 5.46 5.46 NT 2 5.45 5.45 5.47 NT 4 5.46 5.455.45 NT 8 5.45 5.45 5.45 5.46

TABLE 15 pH after storage at +45° C. pH Time (days) CF TF1A TF1B TF1C 05.50 5.52 5.52 5.52 1 5.47 5.48 5.48 NT 2 5.47 5.47 5.46 NT 5 5.48 5.485.47 5.49 NT = not tested

(B) Concentration Analysis

Samples were diluted to an appropriate level with the relevant bufferand their absorbance at 280 nm determined using an HP8453 UV/visiblespectrophotometer (Agilent Technologies). Absorbance values wereconverted to BAK502G9 concentrations using the known extinctioncoefficient of 1.723. The results of absorbance measurement offormulations CF, TF1A, TF1B and TF1C at each temperature are shown inTables 16-20 below:

TABLE 16 BAK502G9 concentration after storage at −70° C. Meanconcentration ± SD^(a) (mg/ml) Time (months) CF TF1A TF1B TF1C 0 10.8 ±0.1 48.9 ± 0.3 107.4 ± 0.9 147.7 ± 4.6 3 11.0 ± 0.1 52.4 ± 0.7 112.8 ±0.6 NT 6 10.3 ± 0.1 46.4 ± 0.6 104.5 ± 2.1 NT 9 10.6 ± 0.2 46.9 ± 0.8105.8 ± 0.7 151.8 ± 1.4 12 10.7 ± 0.1 50.9 ± 0.0 109.3 ± 0.3 152.7

TABLE 17 BAK502G9 concentration after storage at +5° C. Meanconcentration ± SD^(a) (mg/ml) Time (months) CF TF1A TF1B TF1C 0 10.8 ±0.1 48.9 ± 0.3 107.4 ± 0.9 147.7 ± 4.6 3 10.8 ± 0.2 51.4 ± 0.3 110.7 ±0.3 NT 6 10.4 ± 0.2 46.4 ± 0.9  99.1 ± 0.9 NT 9 10.6 ± 0.1 46.6 ± 0.5105.9 ± 0.9 150.2 ± 0.2 12 10.7 ± 0.1 50.5 ± 0.1 112.3 ± 0.4 156.1 ± 0.5

TABLE 18 BAK502G9 concentration after storage at +25° C. Meanconcentration ± SD^(a) (mg/ml) Time (months) CF TF1A TF1B TF1C 0 10.8 ±0.1 48.9 ± 0.3 107.4 ± 0.9 147.7 ± 4.6 3 10.8 ± 0.2 53.8 ± 0.4 116.2 ±0.5 NT 6 10.4 ± 0.1 46.5 ± 0.4 102.9 ± 2.7 NT 9 10.6 ± 0.2 46.2 ± 0.9106.3 ± 1.2 152.3 ± 1.9 12 10.2 ± 0.1 52.0 ± 0.9 110.8 ± 0.5 155.8 ± 1.3

TABLE 19 BAK502G9 concentration after storage at +37° C. Meanconcentration ± SD^(a) (mg/ml) Time (weeks) CF TF1A TF1B TF1C 0 10.4 ±0.1 46.4 ± 0.4  98.6 ± 1.2 142.3 ± 0.8 1 11.1 ± 0.2 52.1 ± 0.7 112.1 ±0.8 NT 2 11.0 ± 0.3 51.4 ± 1.3 118.7 ± 0.3 NT 4 10.9 ± 0.3 54.2 ± 0.4120.6 ± 1.0 NT 8 11.0 ± 0.2 56.2 ± 1.9 117.2 ± 0.2 165.8 ± 1.1

TABLE 20 BAK502G9 concentration after storage at +45° C. Meanconcentration ± SD^(a) (mg/ml) Time (days) CF TF1A TF1B TF1C 0 10.4 ±0.1 46.4 ± 0.4  98.6 ± 1.2 142.3 ± 0.8 1 10.6 ± 0.2 54.6 ± 1.7 120.4 ±2.4 NT 2 10.7 ± 0.1 54.7 ± 1.4 124.9 ± 1.3 NT 5 10.6 ± 0.3 54.7 ± 2.0122.2 ± 0.3 151.5 ± 1.6 ^(a)n = 3 NT = not tested

(C) Gel filtration HPLC Analysis

Gel filtration HPLC was performed on an HP1100 system (AgilentTechnologies). A TSK-Gel 3000S column was equilibrated with 0.2M sodiumphosphate pH7.5. Samples were diluted to 1 mg/ml with the relevantbuffer and centrifuged at 13,000 rpm for 10 minutes to remove anyparticulate matter. 3×20 μl injections of each sample were made onto thecolumn, which was run at a flow rate of 1 ml/min. A variable wavelengthdetector was used to monitor absorbance at 220 and 280 nm.

The results of gel filtration analysis of formulations CF, TF1A, TF1Band TF1C at each temperature are shown in FIGS. 8-12.

(D) Reduced SDS-PAGE Analysis

Samples were diluted to 1 mg/ml with the relevant buffer and 16.70 addedto 12.50 of 4X LDS sample buffer (Invitrogen), 15.8 μl of Milli-Q waterand 5 μl reducing agent (Invitrogen). The samples were heated at 95° C.for one minute and then placed on ice. 15 μl of each sample was loadedonto a 4-12% BisTris gel (Invitrogen) in a tank containing 1× MES SDSrunning buffer and the gel run for 35 minutes at a constant current of500 mA. After electrophoresis the gel was removed from its casing,rinsed for 3×10 minutes with Milli-Q water, stained with Gelcode® Bluestaining reagent (Pierce) for a minimum of one hour and then destainedwith Milli-Q water. The gel was photographed and analysed using a UVPGDS8000 gel documentation system. The relative abundance of BAK502G9heavy and light chain in each sample was determined.

The results of reduced SDS-PAGE analysis of formulations CF, TF1A, TF1Band TF1C at each temperature are shown in FIGS. 13, 15, 17, 19 and 21.Measurements of abundance of BAK502G9 heavy and light chains at eachtemperature are also shown in FIGS. 14, 16, 18, 20 and 22.

(E) Non-Reduced SDS-PAGE Analysis

Samples were diluted to 1 mg/ml with the relevant buffer and 16.7 μladded to 25 μl of 2× non-reducing sample buffer (0.125M Tris pH6.8,4%(w/v) SDS, 30%(v/v) glycerol, 0.004%(w/v) bromophenol blue), 3.3 μl ofMilli-Q water and 5 μl 1M iodoacetamide. The samples were heated at 95°C. for one minute and then placed on ice. 15 μl of each sample wasloaded onto a 4-12% BisTris gel (Invitrogen) in a tank containing 1× MESSDS running buffer and the gel run for 35 minutes at a constant currentof 500 mA. After electrophoresis the gel was removed from its casing,rinsed for 3×10 minutes with Milli-Q water, stained with Gelcode® Bluestaining reagent (Pierce) for a minimum of one hour and then destainedwith Milli-Q water. The gel was photographed and analysed using a UVPGDS8000 gel documentation system. The relative abundance of BAK502G9monomer in each sample was determined.

The results of non-reduced SDS-PAGE analysis of formulations CF, TF1A,TF1B and TF1C at each temperature are shown in FIGS. 23, 25, 27, 29 and31. Measurements of abundance of intact BAK502G9 monomer at eachtemperature are also shown in FIGS. 24, 26, 28, 30 and 32.

(F) Isoelectric Focusing Analysis

Prior to sample loading, the electrophoresis bed was cooled and a pH3-10IEF gel (Cambrex) was prefocused for 10 minutes at 1 W, 2000V, 150 mAusing an Apelex PS9009TX power pack. Samples were diluted to 1 mg/mlwith the relevant buffer. A sample mask was placed on the surface of thegel and 5 μl of each sample was loaded. The gel was prefocused again andthe sample mask removed. The gel was then focused for 60 minutes at 25W, 1500V, 50 mA. After electrophoresis, the gel was fixed with 50% (v/v)methanol, 6% (w/v) trichloroacetic acid, 3.6% (w/v) 5-sulphosalicyclicacid for 30 minutes, then washed with water and dried in an oven at40-50° C. for one hour. The gel was stained for 30 minutes usingPhastGel Blue R (Pharmacia; one tablet dissolved in 60% (v/v) methanol),washed with Milli-Q water to remove excess stain and then destained forapproximately 3 minutes with 9% (v/v) glacial acetic acid, 25% (v/v)ethanol solution. The gel was dried in an oven at 40-50° C. for onehour. The dried gel was photographed and analysed using a UVP GDS8000gel documentation system. The number and pI range of the isoforms ineach sample was determined and the results observed with formulationsCF, TF1A, TF1B and TF1C at each temperature are shown in Tables 21-25below:

TABLE 21 pI range and number of isoforms by IEF after storage at −70° CpI range (number of isoforms) Time (months) CF TF1A TF1B TF1C 06.75-7.17 (4) 6.74-7.17 (4) 6.71-7.14 (4) 6.69-7.10 (4) 3 6.39-6.76 (4)6.39-6.74 (4) 6.38-6.73 (4) NT 6 6.53-6.78 (4) 6.63-6.85 (4) 6.64-6.88(4) NT 9 6.60-6.89 (4) 6.60-6.88 (4) 6.60-6.89 (4) 6.61-6.89 (4) 126.67-6.93 (4) 6.64-6.91 (4) 6.65-6.96 (4) 6.69-6.99 (4)

TABLE 22 pI range and number of isoforms by IEF after storage at +5° C.pI range (number of isoforms) Time (months) CF TF1A TF1B TF1C 06.75-7.17 (4) 6.74-7.17 (4) 6.71-7.14 (4) 6.69-7.10 (4) 3 6.59-6.85 (4)6.59-6.85 (4) 6.59-6.86 (4) NT 6 6.66-6.91 (4) 6.65-6.89 (4) 6.68-6.95(4) NT 9 6.66-6.94 (4) 6.66-6.94 (4) 6.64-6.90 (4) 6.63-6.89 (4) 126.62-6.91 (4) 6.63-6.94 (4) 6.62-6.92 (4) 6.61-6.89 (4)

TABLE 23 pI range and number of isoforms by IEF after storage at +25° C.pI range (number of isoforms) Time (months) CF TF1A TF1B TF1C 06.75-7.17 (4) 6.74-7.17 (4) 6.71-7.14 (4) 6.69-7.10 (4) 3 6.59-6.80 (4)6.62-6.83 (4) 6.61-6.84 (4) NT 6 6.65-6.90 (4) 6.69-6.98 (4) 6.64-6.93(4) NT 9 6.62-6.85 (4) 6.61-6.85 (4) 6.64-6.87 (4) 6.64-6.89 (4) 126.62-6.85 (4) 6.61-6.86 (4) 6.61-6.86 (4) 6.62-6.88 (4)

TABLE 24 pI range and number of isoforms by IEF after storage at +37° CpI range (number of isoforms) Time (weeks) CF TF1A TF1B TF1C 0 6.65-6.95(4) 6.65-6.93 (4) 6.63-6.92 (4) 6.64-6.93 (4) 1 6.47-6.77 (4) 6.60-6.84(4) 6.63-6.88 (4) NT 2 6.57-6.78 (4) 6.56-6.79 (4) 6.58-6.82 (4) NT 46.62-6.84 (4) 6.68-6.95 (4) 6.69-6.98 (4) NT 8 6.61-6.86 (5) 6.20-6.85(7) 6.28-6.89 (7) 6.26-6.91 (7)

TABLE 25 pI range and number of isoforms by IEF after storage at +45° C.pI range (number of isoforms) Time (days) CF TF1A TF1B TF1C 0 6.65-6.95(4) 6.65-6.93 (4) 6.63-6.92 (4) 6.64-6.93 (4) 1 6.61-6.84 (4) 6.64-6.88(4) 6.64-6.89 (4) NT 2 6.60.6.85 (4) 6.56-6.78 (4) 6.62-6.85 (4) NT 56.63-6.91 (4) 6.65-6.95 (4) 6.64-6.95 (4) 6.64-6.93 (4) NT = not tested

Summary of Stability Analysis Results

Stability at −70° C.

Both CF and TF1A were stable for 12 months at −70° C. The analyticalprofiles were similar at 0 and 12 months except for the % intact IgG bynon-reduced SDS-PAGE, which decreased from 95.9% at t=0 to 89.9% at 12months for CF and from 96.2% at t=0 to 89.2% at 12 months for TF1A. ByHPLC after 12 months, the percentage monomer for both CF and TF1Asamples was 100%. Both CF and TF1A samples displayed 4 bands on IEFafter 12 months. The results indicated that CF and TF1A are comparableat this temperature.

Stability at 5° C.

Both CF and TF1A were stable for 12 months at 5° C. The analyticalprofiles were similar at 0 and 12 months except for the % intact IgG bynon-reduced SDS-PAGE, which decreased from 95.9% at t=0 to 89.5% at 12months for CF and from 96.2% at t=0 to 88.9% at 12 months for TF1A. ByHPLC after 12 months, the percentage monomer for both CF and TF1Asamples was 100%. Both CF and TF1A samples displayed 4 bands on IEFafter 12 months. The results indicated that CF and TF1A are comparableat this temperature.

Stability at 25° C.

Both CF and TF1A were stable for 12 months at 25° C. The % intact IgG bynon-reduced SDS-PAGE decreased from 95.9% at t=0 to 89.1% at 12 monthsfor CF and from 96.2% at t=0 to 87.5% at 12 months for TF1A. There isalso a minor high molecular weight (>220 kDa) band in both formulationson the non-reduced SDS-PAGE PAGE gel that was not detected at t=0. ByHPLC after 12 months, the percentage monomer for both CF and TF1Asamples were 98.9 and 96.42%, respectively. Both CF and TF1A samplesdisplayed 4 bands on IEF after 12 months. The results indicated that CFand TF1A are comparable at this temperature.

Stability at 37° C.

Both CF and TF1A were stable for 4 weeks at 37° C. but failed to meetthe draft specification for some parameters after 8 weeks, namely purityby reduced SDS-PAGE (both formulations) and % monomer by GF-HPLC (TF1Aonly; borderline result). The results indicated that CF is more stablethan TF1A at this temperature over an 8 week period.

Stability at 45° C.

Both CF and TF1A were stable for 5 days at 45° C. although there wereslight changes in the analytical profiles after this time (someadditional minor bands on SDS-PAGE and IEF gels). The results indicatedthat CF and TF1A are comparable at this temperature over a 5 day period.

1. A pharmaceutical composition comprising an IL-13 antibody and between10 and 200 mM sodium chloride, between 0.001 and 0.1% (w/w) Polysorbate80, and between 1 and 100 mM sodium acetate, at a pH between 5.2 to 5.7.2. A pharmaceutical composition as defined in claim 1 wherein the IL-13antibody is a human IL-13 monoclonal antibody.
 3. A pharmaceuticalcomposition as defined in claim 1 wherein the IL-13 antibody is presentwithin the pharmaceutical composition in an amount of between 1 and 200mg/ml. 4-12. (canceled)
 13. A pharmaceutical composition as defined inclaim 1 which comprises 50 mg/ml of an IL-13 antibody, 85 mM sodiumchloride and 0.01% (w/w) Polysorbate 80 buffered to a pH of 5.5±0.1 with50 mM sodium acetate buffer. 14-20. (canceled)
 21. Use of apharmaceutical antibody composition of claim 13 in the manufacture of amedicament for the treatment of an IL-13 related disorder.
 22. Use asdefined in claim 21 wherein the IL-13 related disorder is selected fromasthma, atopic dermatitis, allergic rhinitis, fibrosis, chronicobstructive pulmonary disease, scleroderma, inflammatory bowel diseaseand Hodgkin's lymphoma.
 23. Use as defined in claim 22 wherein the IL-13related disorder is asthma.
 24. A method of treatment or prophylaxis ofan IL-13 related disorder which comprises administering to the sufferera therapeutically effective amount of a pharmaceutical antibodycomposition as defined in claim
 1. 25. A pharmaceutical antibodycomposition as defined in claim 1 for use in the treatment of an IL-13related disorder.